Thursday, April 24, 2025
4/24/2025
Molecular Analysis of Flagellar Dynein Function - Motile cilia and flagella play key roles in development, fertility, and organismal homeostasis; in humans, defects result in a broad array of phenotypes such as male/female infertility, hydrocephalus, severe bronchial problems and heart malformations. Cilia contain more than 700 distinct protein components and indeed, more than 5% of all human genes are involved in the assembly or function of these motile/sensory organelles. Ciliary motility is powered by the highly complex inner and outer dynein arm motors whose activity results in inter-doublet microtubule (MT) sliding and ciliary beating. However, the molecular mechanisms by which dyneins and other ciliary subsystems are pre-assembled in cytoplasm, docked at specific axonemal locations, and how their activity is controlled by the mechanical state or curvature of the axoneme to generate and propagate specific waveforms remain very unclear. In this proposal we will address key aspects of these fundamental problems in ciliary biology using two model organisms with very complementary attributes: Chlamydomonas will be used for genetic/biochemical and structural approaches, whereas RNAi methods in planaria will be employed to assess the function of novel factors in the context of a ciliated epithelium where thousands of motile cilia are synchronized through hydrodynamic coupling. We recently found that a WD-repeat protein (WDR92), which interacts with a prefoldin-like co-chaperone complex, is necessary to build fully functional motile cilia; lack of WDR92 results in axoneme assembly defects including missing dynein arms, incomplete outer doublet MTs and failure of the central pair complex to form. In Aim 1 we will use biochemical methods in Chlamydomonas to identify WDR92-interacting components in cytoplasm and then test their role in ciliary formation and function in planaria, as this will provide new paradigms for understanding how cytoplasmic factors influence the coordinate assembly of axonemal substructures. Once trafficked into the ciliary compartment, assembling outer arm dyneins at precise locations is a multi-factorial process that requires both specific docking proteins within the axonemal superstructure and soluble components in the ciliary matrix. In Aim 2, we will use biochemical/structural methods to define the mechanistic roles of two essential components in the precisely patterned assembly of the outer dynein arm that is absolutely critical for building a fully functional organelle. Axonemal dyneins must sense and respond to the curvature that they experience in order for regions of active sliding to oscillate across the structure and to propagate a wave of motor activity along the organelle generating a ciliary beat. We have predicted that the leucine-rich repeat protein LC1 which binds MTs and also associates with the MT-binding domain of one dynein heavy chain is key to this mechano-switching. In Aim 3, we will use a newly available LC1 null mutant to rigorously test these mechanistic hypotheses by expressing mutant versions of LC1 designed based on our biochemical/NMR structural studies. This will provide direct mechanistic insight into a conserved dynein regulatory system that is fundamental to the generation and propagation of ciliary beats.
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